Aim To test the anti-inflammatory biological activity of prokaryotic expressed lunasin, and to lay the foundation for future lunasin development in biomedical field. Methods The lunasin DNA fragment amplified by PCR was cloned into the expression vector pET28 by means of genetic engineering technology. After the identification by restriction enzyme digestion and sequencing, lunasin expression was induced. The purification of lunasin was performed by nickel ion affinity chromatography. Griess method and ELISA were used to test the effects of lunasin on the contents of nitric oxide, tumor necrosis factor-α and interleukin-6 in the culture medium of mouse J774A.1 cells. The effects of lunasin on the phosphorylation of nuclear factor-κB (NF-κB) p65 and the expression of tissue factor (TF) were detected by Western blot. Results Prokaryotic expression vector of lunasin was successfully constructed and the recombinant lunasin peptide with about 90% purification was obtained. Lunasin could inhibit activation of NF-κB induced by lipopolysaccharide (LPS) in mouse J774A.1 cells and then subsequently suppressed the synthesis of nitric oxide and the expression of downstream genes like tumor necrosis factor-α and interleukin-6. Lunasin also significantly inhibited the expression of TF induced by LPS in J774A.1 cells. Conclusion Prokaryotic expressed lunasin shows significant anti-inflammatory biological activity, and significantly inhibits the TF expression induced by LPS in J774A.1 cells, and its mechanism may be achieved by inhibiting the activation of NF-κB.